PROJECT SUMMARY Histidine phosphorylation in mammalian cell signaling is largely unexplored despite the biological importance where few phosphohistidine (pHis) targets have been identified and validated. These critical biological targets include proteins involved in fatty acid biosynthesis, cell signaling, regulation of gene transcription and ion transport. Given the importance of these fundamental cellular processes and related impact of histidine phosphorylation on protein function, dysregulation of pHis levels has been implicated as a mechanism underlying the pathophysiology of disease including liver disease, which is among the top 15 leading causes of death in the United States. Alcoholic liver disease (ALD) accounts for approximately 50% of these deaths. Elucidation of the mechanisms underlying the pathophysiology of ALD is critical to ultimately developing novel clinical interventional therapy, particularly at early onset. Our research group has been actively investigating the role of a mammalian phosphohistidine phosphatase, PHPT1, in ethanol-induced hepatocyte dysfunction and our data has demonstrated the following critical results. First, we have identified that PHPT1 is significantly downregulated and cytoplasmic pHis levels are increased in the livers of chronic ethanol-exposed mice. Second, our in vivo data has demonstrated that transgenic delivery of PHPT1 inhibits hepatic steatosis in ethanol-fed mice. Taken together, we hypothesize that chronic ethanol exposure induces downregulation of PHPT1 and subsequent changes in hepatic pHis signaling, altering expression and activity of critical hepatic lipogenic targets contributing to the onset of hepatic steatosis. To address this, we have generated a novel PHPT1 knockout mouse. Our initial evidence indicates disruption of PHPT1 is embryonically lethal. Our heterozygote PHPT1 mice have an approximate 50% reduction of PHPT1, particularly in the liver but further characterization is needed. Therefore, the purpose of this R03 is to fully examine the steatotic phenotype of the PHPT1 knockout in relation to PHPT1 inhibition of ethanol-induced hepatic steatosis. Central to addressing our hypothesis, the PHPT1 KO will be exposed to the NIAAA chronic binge model with 2 specific aims: (1) Determine if the reduction of PHPT1 promotes ethanol-induced hepatic steatosis in the PHPT1 Knockout (PHPT1KO) mouse and (2) Determine if liver-specific expression of PHPT1 can rescue hepatic steatosis in PHPT1+/-. The proposed studies will significantly contribute to the understanding of ALD pathophysiology. Our primary scientific goals of this proposal are strongly suited to the mission of NIAAA and that of the R03 small grant program. Our self-contained research project already has both an established essential animal model (PHPT1KO) and additional tools (Ad-PHPT1) to rapidly employ to directly address the role of PHPT1 in ethanol-induced hepatic steatosis.